Drug-receptor models are the basic framework by which drug actions are understood. Our interest has focused on the cardiac glycosides, among the ten most-prescribed drugs today. They are very important in the control and prophylaxis of cardiac dysrhythmias, as well as for their more emphasized cardiotonic effects. The physiological receptor for these steroids is generally believed to be Na+,K+-ATPase, an enzyme with crucial roles in maintaining cell homeostasis. We have found an apparently universal relationship between the C17 carbonyl oxygen position of digitalis drugs and biological activity. This correlation was the basis of a successful effort to design a covalent binding probe to specifically label the corresponding site on Na+,K+=ATPase. The main aim of the continuing study will be to determine the amino acid composition and sequence of the site using this probe. A second aim will be to answer fundamental questions about the structural, conformational and electronic basis of the carbonyl oxygen relationship. In particular, we will: 1) study association and dissociation kinetics with 3H-labeled glycosides varying in carbonyl oxygen position; 2) test existing drug-receptor binding models by synthesizing and studying analogs with restricted side group rotation and electronically modified C17 side groups; 3) use the MM2p program to study side group rotation and calculate dipole moments; and 4) calculate electrostatic potentials of model side groups. A series of glycosides stepwise varying in structure has yielded surprising results unexplained by current drug-receptor models. Our approach will be the same we used to discover the key role of carbonyl oxygen position--a unique combination of synthesis of selected glycosides, biological studies with Na+,K+-ATPase and cat heart muscle preparations, X-ray crystallography (basis of precise atomic positions for conformational studies and computer graphics), and molecular mechanics. Two NIH compute resources are central to the research: PROPHET and MMS-X.